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ABSTRACT: Mesd is a specialized chaperone for low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6. In our previous studies, we found that Mesd binds to mature LRP6 on the cell surface and blocks the binding of Wnt antagonist Dickkopf-1 (Dkk1) to LRP6. Herein, we demonstrate that Mesd also binds to LRP5 with a high affinity and is a universal inhibitor of LRP5 and LRP6 ligands. Mesd not only blocks binding of Wnt antagonists Dkk1 and Sclerostin to LRP5 and LRP6 but also inhibits Wnt3A and Rspondin1-induced Wnt/beta-catenin signaling in LRP5- and LRP6-expressing cells. We also found that Mesd, Dkk1, and Sclerostin compete with one another for binding to LRP5 and LRP6 at the cell surface. More importantly, we demonstrated that Mesd is able to suppress LRP6 phosphorylation and Wnt/beta-catenin signaling in prostate cancer PC-3 cells and inhibits PC-3 cell proliferation. Our results indicate that recombinant Mesd protein is a useful tool for studying Wnt/beta-catenin signaling on the cell surface and has a potential therapeutic role in Wnt-dependent cancers.
Biochemistry 06/2010; 49(22):4635-43. · 3.42 Impact Factor
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ABSTRACT: Cks1 plays an essential role in SCFSkp2-mediated ubiquitination, and consequently turnover, of the cdk2 inhibitor and tumor supressor p27Kip1. High Cks1 expression is associated with aggressive breast tumors and correlates with low p27Kip1 levels in some cases, although it is also an independent prognostic marker for survival, and provides predictive information in addition to that provided by p27Kip1 alone. In this report we demonstrate that Cks1 protein and mRNA are elevated to very high levels in mammary tumors initiated by erbB2, c-myc and polyoma middle-T (PyMT) in transgenic mice, whereas Cks1 protein is hardly detectable in the normal mammary epithelium. Cks1 is also highly upregulated in rat mammary tumors initiated by methylnitrosourea (MNU). Despite high levels of Cks1 expression, p27Kip1 levels were not reduced, and were in fact slightly higher in mammary tumors initiated by erbB2, PyMT and MNU. In contrast mammary tumors from MMTV-c-myc mice did exhibit low p27Kip1 and higher levels of Skp2. Together, these data suggest that deregulated Cks1 expression might play roles in oncogene and carcinogen-initiated mammary tumorigenesis independent of p27Kip1 turnover in certain tumors. Stable overexpression of Cks1 in human breast carcinoma MCF-7 cells did not significantly reduce p27Kip1 expression, although it conferred resistance to Faslodex (ICI 182780)-mediated inhibition of colony outgrowth in these cells. In contrast, Cks1-depleted MCF-7 cells formed fewer colonies in estrogen-containing medium. Therefore, our studies also suggest that Cks1 levels regulate the responsiveness of ER+ breast cancers to estrogens and anti-estrogens.
International Journal of Oncology 06/2009; 34(5):1425-31. · 2.40 Impact Factor
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Zhican Qu,
Sabrina Van Ginkel,
Anshu M Roy,
Louise Westbrook,
Mubina Nasrin,
Yulia Maxuitenko,
Andra R Frost,
Delicia Carey,
Wenquan Wang,
Rongbao Li,
William E Grizzle, Jaideep V Thottassery,
Francis G Kern
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ABSTRACT: Clinical studies have shown that decreased tamoxifen effectiveness correlates with elevated levels of vascular endothelial growth factor (VEGF)-A(165) in biopsy samples of breast cancers. To investigate the mechanisms underlying tamoxifen resistance and metastasis, we engineered the estrogen receptor (ER)-positive MCF-7 human breast cancer cell line to express VEGF to clinically relevant levels in a doxycycline-regulated manner. Induction of VEGF expression in orthotopically implanted xenografts that were initially tamoxifen responsive and noninvasive resulted in tamoxifen-resistant tumor growth and metastasis to the lungs. Lung metastases were also observed in a VEGF-dependent manner following tail vein injection of tumor cells. At both primary and metastatic sites, VEGF-overexpressing tumors exhibited extensive fibroblastic stromal content, a clinical feature called desmoplasia. VEGF-induced metastatic colonies were surrounded by densely packed stromal cells before detectable angiogenesis, suggesting that VEGF is involved in the initiation of desmoplasia. Because expression of VEGF receptors R1 and R2 was undetectable in these tumor cells, the observed VEGF effects on reduction of tamoxifen efficacy and metastatic colonization are most likely mediated by paracrine signaling that enhances tumor/stromal cell interactions and increases the level of desmoplasia. This study reveals new roles for VEGF in breast cancer progression and suggests that combination of antiestrogens and VEGF inhibitors may prolong tamoxifen sensitivity and prevent metastasis in patients with ER-positive tumors.
Cancer Research 09/2008; 68(15):6232-40. · 7.86 Impact Factor
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ABSTRACT: Cks1, a small protein whose expression is strongly associated with aggressive breast tumors, is a component of cyclin-cdk complexes, as well as the SCF(Skp2) ubiquitin ligase. In these studies, we explored its roles in estrogen receptor-positive breast tumor cells. When exposed to the antiestrogen ICI 182780, these cells accumulate in G(1) by reducing the expression of Cks1, and increasing the levels of p130/Rb2, a cdk2 inhibitor and SCF(Skp2) target. Heregulin beta1 or estradiol abrogate antiestrogen effects by increasing Cks1 expression, down-regulating p130/Rb2 and inducing S phase entry. Depletion of Cks1 in these cells by RNA interference concomitantly decreased Skp2 and up-regulated p130/Rb2 and another SCF(Skp2) target, p27(Kip1). Remarkably, however, Cks1-depleted cells not only exhibit slowed G(1) progression, but also accumulate in G(2)-M due to blocked mitotic entry. Notably, we show that cdk1 expression, which is crucial for M phase entry, is drastically diminished by Cks1 depletion, and that restoration of cdk1 reduces G(2)-M accumulation in Cks1-depleted cells. cdk1 reduction in Cks1-depleted cells is a consequence of a marked decrease in its mRNA and not due to alteration in its proteolytic turnover. Both heregulin beta1 and estradiol could neither restore cdk1 nor sustain cycling in Cks1-depleted cells, although classical estrogen receptor function remained unaltered. Cks1 depletion also decreased Skp2 in human mammary epithelial cells without altering cell cycle progression. Thus, the indispensability of Cks1 to the breast cancer cell cycle, versus its redundancy in normal cells, suggests that Cks1 abrogation could be an effective interventional strategy in breast cancer.
Cancer Research 01/2008; 67(23):11393-401. · 7.86 Impact Factor
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ABSTRACT: The role of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling in estrogen receptor positive (ER(+)) MCF-7 breast carcinoma cells is not well understood. We depleted MEK by cotransfection of MEK1 and MEK2 siRNA duplexes in a MCF-7 derived line (MCF-7/ lacZ, ML-20) and determined its effect on serum, 17beta-estradiol (E(2)), and growth factor induced DNA synthesis. MEK knockdown did not decrease fetal bovine serum-induced DNA synthesis in ML-20 cells although it did inhibit DNA synthesis induced by estrogen-stripped calf serum (CCS) suggesting that MEK activation plays an important role in growth signaling induced by serum components other than estrogen. Consistent with this notion, MEK knockdown only modestly decreased DNA synthesis induced by E(2)-supplemented CCS medium in ML-20 cells. Similarly, MEK knockdown only caused moderate decreases in DNA synthesis induced by fibroblast growth factor-1 (FGF-1) or heregulin-beta1 (HRGbeta1) in this media. Also, there were only minimal effects of MEK knockdown in cells treated with growth factor-supplemented serum-free medium. Although MEK depletion inhibited ERK1/2 phosphorylation induced by CCS in these cells, that induced by growth factor supplemented CCS media was relatively unaffected. Similarly, ERK1/2 phosphorylation induced by growth factor-supplemented serum-free media was also relatively unaffected by MEK depletion. These results suggest that pathways regulating DNA synthesis induced by serum in MCF-7 cells are significantly more dependent on constitutive MEK levels than that induced by E(2) or growth factors.
International Journal of Oncology 01/2007; 29(6):1573-80. · 2.40 Impact Factor
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ABSTRACT: Supplementation with exogenous growth factors such as fibroblast growth factors (FGFs) is essential for anchorage-independent growth of the SW-13 human adrenal adenocarcinoma cell line. We have found that SW-13 cells express mRNAs for FGFRs 1, 3, and 4, but not FGFR2. To assess the roles of individual FGFRs, in anchorage-independent growth, we determined the effects of down-regulation of each FGFR on FGF2- and FGF4-mediated soft agar colony formation in these cells. Using RNAi strategies we found that knockdown of either FGFR1 or FGFR3 leads to inhibition of FGF2- or FGF4-induced soft agar clonogenicity without affecting that induced by heregulin beta1. However, this inhibition is independent of ERK1/2 activation as levels of FGF-induced phospho-ERK 1/2 remain unchanged upon knockdown of either FGFR1 or FGFR3. Conversely, RNAi-mediated knockdown of FGFR4 appeared to have no significant effect on either FGF2- or FGF4-induced anchorage-independent colony formation, or ERK1/2 phosphorylation. These results suggest that constitutive levels of both FGFR1 and FGFR3, but not FGFR4 are essential for FGF-stimulated anchorage-independent growth of SW-13 cells.
Oncology Reports 07/2006; 15(6):1407-16. · 1.84 Impact Factor
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ABSTRACT: Nucleoside anticancer drugs like gemcitabine (2'-deoxy-2',2'-difluorocytidine) are potent inducers of p53, and ectopic expression of wild-type p53 sensitizes cells to these agents. However, it is also known that nucleosides are efficient activators of apoptosis in tumor cells that do not express a functional p53. To clarify this issue, we examined the effects of gemcitabine and 4'-thio-beta-d-arabinofuranosylcytosine (T-ara-C) on p73, a structural and functional homologue of p53, whose activation could also account for nucleoside-induced apoptosis because no functionally significant mutations of p73 have been reported in cancers. Acute treatment of HCT 116 colon carcinoma cells with gemcitabine or T-ara-C induced marked cytotoxicity and cleavage of caspase-3 and poly(ADP-ribose) polymerase. T-ara-C and gemcitabine markedly induced p53 accumulation as well as increased levels of phospho-p53 (Ser15/Ser20/Ser46) and induced its binding to a consensus p53 response element. Despite robust activation of p53 by T-ara-C and gemcitabine, we found that wild-type and p53-/- HCT 116 cells exhibited almost equivalent sensitivity towards these nucleosides. Examination of p73 revealed that T-ara-C and gemcitabine markedly increased p73 protein levels and p73 DNA-binding activities in both p53-/- and wild-type cells. Furthermore, T-ara-C- and gemcitabine-induced increases in p73 levels occur due to a decrease in p73 protein turnover. RNA interference studies show that nucleoside-induced p73 increases are independent of c-Abl, a nucleoside-activated kinase recently implicated in p73 stabilization. HCT 116 lines, wherein the downstream p53/p73 targets Bax and PUMA (p53 up-regulated modulator of apoptosis) were deleted, were less sensitive to T-ara-C and gemcitabine. Together, these studies indicate that c-Abl-independent p73 stabilization pathways could account for the p53-independent mechanisms in nucleoside-induced apoptosis.
Molecular Cancer Therapeutics 03/2006; 5(2):400-10. · 5.23 Impact Factor
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ABSTRACT: Stromal fibroblasts associated with in situ and invasive breast carcinoma differ phenotypically from fibroblasts associated with normal breast epithelium, and these alterations in carcinoma-associated fibroblasts (CAF) may promote breast carcinogenesis and cancer progression. A better understanding of the changes that occur in fibroblasts during carcinogenesis and their influence on epithelial cell growth and behavior could lead to novel strategies for the prevention and treatment of breast cancer. To this end, the effect of CAF and normal breast-associated fibroblasts (NAF) on the growth of epithelial cells representative of pre-neoplastic breast disease was assessed.
NAF and CAF were grown with the nontumorigenic MCF10A epithelial cells and their more transformed, tumorigenic derivative, MCF10AT cells, in direct three-dimensional co-cultures on basement membrane material. The proliferation and apoptosis of MCF10A cells and MCF10AT cells were assessed by 5-bromo-2'-deoxyuridine labeling and TUNEL assay, respectively. Additionally, NAF and CAF were compared for expression of insulin-like growth factor II as a potential mediator of their effects on epithelial cell growth, by ELISA and by quantitative, real-time PCR.
In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells. However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells. The degree of growth inhibition varied among NAF or CAF from different individuals. In greater numbers, NAF and CAF have less inhibitory effect on epithelial cell growth. The rate of epithelial cell apoptosis was not affected by NAF or CAF. Mean insulin-like growth factor II levels were not significantly different in NAF versus CAF and did not correlate with the fibroblast effect on epithelial cell proliferation.
Both NAF and CAF have the ability to inhibit the growth of pre-cancerous breast epithelial cells. NAF have greater inhibitory capacity than CAF, suggesting that the ability of fibroblasts to inhibit epithelial cell proliferation is lost during breast carcinogenesis. Furthermore, as the degree of transformation of the epithelial cells increased they became resistant to the growth-inhibitory effects of CAF. Insulin-like growth factor II could not be implicated as a contributor to this differential effect of NAF and CAF on epithelial cell growth.
Breast cancer research: BCR 02/2005; 7(1):R46-59. · 5.24 Impact Factor
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ABSTRACT: Increased growth factor receptor signaling is implicated in antiestrogen-resistant breast tumors suggesting that abrogation of such signaling could restore or prolong sensitivity to antihormonal agents. Activation of the mitogen-activated protein/extracellular regulated kinase kinase (MEK)-extracellular regulated kinase (ERK)1/2 cascade is a common component of such pathways. We investigated the ability of the MEK activation inhibitor U0126 to block the increased growth of estrogen receptor-positive MCF-7 breast cancer cells caused by fibroblast growth factor 1 (FGF-1), heregulin beta1 (HRGbeta1), and epidermal growth factor (EGF) in the presence of the pure antiestrogen ICI 182780 (Faslodex; fulvestrant). We found that either FGF-1 or HRGbeta1 but not EGF substantially reduced the inhibitory effects of U0126 on growth and ERK1/2 activation, including the combined inhibitory effects of U0126 and ICI 182780. FGF-1 and HRGbeta1 also reduced the inhibition of ERK1/2 phosphorylation by the MEK inhibitors PD98059 and PD184161. Interestingly, a transiently transfected dominant-negative MEK1 completely abrogated activation of a coexpressed green fluorescent protein-ERK2 reporter by all three of the factors. Despite a short-lived activation of Ras and Raf-1 by all three of the growth factors, both FGF-1 and HRGbeta1, unlike EGF, induced a prolonged activation of MEK and ERK1/2 in these cells. Thus, activation of FGF-1- and HRGbeta1-specific signaling causes MEK-dependent prolonged activation of ERK1/2, which is incompletely susceptible to known MEK inhibitors. We also demonstrate that the cytosolic phospholipase A2 inhibitor arachidonyl trifluoro methyl ketone and the pan PKC inhibitor bisindolymaleimide abrogated U0126-resistant phosphorylation of ERK1/2 induced by HRGbeta1 but not by FGF-1. Phosphorylation of ERK5 by all three of the factors was also resistant to U0126 suggesting that its activation is not sufficient to overturn growth inhibition due to diminished ERK1/2 activation. Therefore, therapy combining antiestrogens and MEK inhibitors may be ineffective in some antiestrogen-resistant estrogen receptor-positive breast cancers.
Cancer Research 08/2004; 64(13):4637-47. · 7.86 Impact Factor
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ABSTRACT: Inducible expression of tetracycline responsive element (TRE)-regulated genes in nearly all cells in a stable clone has generally been problematic, especially in long-term culture. Heterogeneity of tet-inducible expression is generally attributed to the instability of the original tet-transactivators tTA and rtTA. These transactivators have cryptic splice sites, prokaryotic codons and full VP16 domains, all of which contribute to their instability. Moreover, they also require high concentrations of Doxycycline (Dox). The 5 amino acid substitutions in the rtTA variant rtTA2S-M2 confer exquisite sensitivity to Dox. Moreover, humanized codons, removal of cryptic splice sites and minimal VP16 domains in rtTA2S-M2 results in its being better tolerated within cells. However, the ability of this modified transactivator to maintain homogeneous inducibility in long-term culture has not been examined. We demonstrate that rtTA2S-M2 expressing clones exhibit functional transactivator activity for over 7 months in culture. Furthermore, rtTA2S-M2 expressing clones with chromosomally integrated copies of a TRE-green fluorescent protein (GFP) reporter also exhibited homogeneous inducibility in long-term culture. Importantly, the inherent reduced toxicity and improved stability of rtTA2S-M2 obviates the need to continuously select for its message, once clones with functional transactivator are isolated. The use of rtTA2S-M2 did not, however, preclude clones with stably integrated TRE-reporter from exhibiting leakiness. However, inclusion of flanking double copies of a 'minimal core element' of the chicken beta-globin gene insulator, instead of the 1.4 kb region, in the TRE-reporter was sufficient to markedly reduce the frequency of clones with high basal expression. Inclusion of the insulator core also did not affect the maximal expression levels of the inducible gene, which typically equaled or exceeded that observed with the strong constitutive CMV promoter. Finally, with this system homogeneous inducibility was observed rapidly and with low doses of Dox.
Gene 03/2004; 327(1):61-73. · 2.34 Impact Factor
